The host defense peptide LL‐37 is internalized by human periodontal ligament cells and prevents LPS‐induced MCP‐1 production

Abstract Objective The human host defense peptide LL‐37 both shows antimicrobial effects and modulates host cell properties. Here, we assess the effects of synthesized LL‐37 on lipopolysaccharide (LPS)‐induced inflammation in human periodontal ligament (PDL) cells and investigates underlying mechanisms. Background LL‐37 has been detected in the periodontal tissues, but its functional importance for PDL cell innate immune responses is not known. Methods Human PDL cells were obtained from premolars extracted on orthodontic indications. Cellular pro‐inflammatory monocyte chemoattractant protein‐1 (MCP‐1) mRNA expression was determined using quantitative real‐time RT‐PCR. MCP‐1 protein production was assessed by western blot and ELISA. Internalization of LL‐37 by PDL cells was visualized by immunocytochemistry. Nuclear factor kappa‐light‐chain‐enhancer of activated B‐cell (NF‐κB) activity was assessed by western blot of phosphorylated p65, phosphorylated p105, and IκBα proteins. Binding of LL‐37 to PDL cell DNA was determined by isolation and purification of DNA and dot blot for LL‐37 immunoreactivity. Results Treatment with LL‐37 (1 µmol/L) for 24 hours prevented LPS‐induced stimulation of MCP‐1 expression analyzed both on transcript and on protein levels. Stimulation with LL‐37 (1 µmol/L) for 24 hours had no effect on toll‐like receptor (TLR)2 and TLR4 transcript expression, suggesting that LL‐37 acts downstream of the TLRs. Preincubation with LL‐37 for 60 minutes followed by stimulation with LPS for 24 hours in the absence of LL‐37 completely prevented LPS‐evoked MCP‐1 transcript expression, implying that LL‐37 acts intracellularly and not via binding and neutralization of LPS. In PDL cells stimulated with LL‐37 for 60 minutes, the peptide was internalized as demonstrated by immunocytochemistry, suggesting an intracellular mechanism of action. LL‐37 immunoreactivity was observed both in the cytosol and in the nucleus. Downregulation of LPS‐induced MCP‐1 by LL‐37 was not mediated by reduction in NF‐κB activity as shown by unaltered expression of phosphorylated p65, phosphorylated p105, and IκBα NF‐κB proteins in the presence of LL‐37. Immunoreactivity for LL‐37 was observed in PDL cell DNA treated with but not without 0.1 and 1 µmol/L LL‐37 for 60 minutes in vitro. Conclusion LL‐37 abolishes LPS‐induced MCP‐1 production in human PDL cells through an intracellular, NF‐κB‐independent mechanism which probably involves direct interaction between LL‐37 and DNA.